WO1995011295A1 - An improved process to recover an aqueous fermentation broth - Google Patents
An improved process to recover an aqueous fermentation broth Download PDFInfo
- Publication number
- WO1995011295A1 WO1995011295A1 PCT/EP1994/003510 EP9403510W WO9511295A1 WO 1995011295 A1 WO1995011295 A1 WO 1995011295A1 EP 9403510 W EP9403510 W EP 9403510W WO 9511295 A1 WO9511295 A1 WO 9511295A1
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- Prior art keywords
- fermentation broth
- organic solvent
- water
- broth
- fermentation
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
Definitions
- the present invention relates to a process for recovery of a fermentation product from an aqueous fermentation broth and in particular to modification of the fermentation broth so as to improve filterability.
- This modification can, for instance, be advantageously applied to a fermentation broth of a clavulan ⁇ ic acid producing microorganism to improve the filterability of said fermentation broth and subsequent recovery of clavulanic acid, preferably as a salt.
- Clavulanic acid is an antibiotic and a potent ?-lactamase inhibitor which finds widespread use as a therapeutic agent. It may be employed synergistically with penicillins and cephalos- porins, as described, for instance, in GB patent 1,508,977. It can be produced by fermentation of a clavulanic acid producing microorganism, for instance Streptomyces clavuliqerus, after which a recovery process is required. In published European patent application EP-A-387178, it .has been suggested to use flocculating agents to improve the filterability of the broth. The present invention provides a simpler and cheaper method to improve the filterability of the broth.
- the present invention provides a process for the recovery of a fermentation product from a fermentation broth which includes the addition of a water-miscible organic solvent to the fermentation broth so as to improve the filterability of the same.
- a process is applied to recover clavulanic acid as a salt e.g. the potassium salt, from a fermentation broth of a clavulanic acid producing microorganism, preferably Streptomyces clavuli erus.
- Preferred organic solvents for addition to a fermentation broth for the purpose of a process according to the invention are methanol, ethanol, propanol, isopropanol, acetone and mixtures thereof. Acetone is particularly preferred.
- a suitable amount of water-miscible organic solvent which when added to a fermentation broth will improve filterability is between 10-200% by weight of the initial broth, preferably 50-175% and more preferably 100-140%.
- Such a solvent addition may, for example, be advantageously applied for the preparation of potassium clavulanate in a procedure comprising:
- ⁇ optionally recrystallizing and isolating the potassium clavulanate by filtering and drying.
- acetone is used as the organic solvent for addition to the fermentation broth.
- a suitable potassium salt for use in such a procedure is potassium 2-ethylhexanoate.
- the amine N,N,N' ,N'-tetramethyl-l,2-diaminoethane (TMEDA) is employed, preferably under conditions such that mono- TMEDA clavulanate is formed, as described in PCT application 094/22873.
- Addition of a water-miscible organic solvent to such a fermentation broth may not only improve the filterability of the broth, but may also improve the phase separation rate in the subsequent extraction step. It may additionally be found advan ⁇ tageous to supplement the organic solvent extraction system with a small amount of a de-emulsifier (see the example) .
- an organic solvent to the fermentation broth with gentle acidification, preferably to pH 3-5 by using mineral or organic acids, suitably hydrochloric acid, sulphuric acid or nitric acid. It is also permissible to add a flocculating agent, preferably a non-ionic or anionic agent.
- a fermentation broth obtained in conventional manner by fermentation using S. clavulicrerus is treated with a water-miscible cold solvent, preferably acetone, at 10-200% by weight of the broth, preferab ⁇ ly 50-175% by weight and more preferably 100-140% by weight.
- the solvent added has a temperature of 0-30°C, preferably 3-6°C.
- the water-miscible organic solvent may contain up to 10% by weight of water.
- the resulting mixture is cooled, if necessary, prefer ⁇ ably down to 0-30°C, and most favourably down to 3-6°C.
- the cold mixture is filtered, preferably after addition of a filter aid (e.g. Dicalite 4108) , to separate the biomass including precipitated solids from the aqueous phase, and the resulting filtrate is kept at low temperature, preferably at 0-30°C, and most favourably at 3-6'C.
- a filter aid e.g. Dicalite 4108
- the filtrate is subsequently distilled in conventional manner to remove much of the water-miscible solvent.
- the tem- perature of the concentrate during evaporation is kept at 10-50°C, favourably 15-40°C, and most preferably at 20-30°C.
- the concentration of water-miscible solvent is in the range of 2.5-50% by weight of the total solution, prefer ⁇ ably 4-30%, and most favourably in the range of 10-20%.
- the aqueous solution is further concentrated to increase the concentration of clavulanic acid.
- concentrations of clavulanic acid in the concentrate are 1-50 times the concentration of the broth after fermentation, and more favourably 4-10 times.
- the temperature of the concentrate during the evaporation is kept at 10-50°C, favourable 15-40°C and most preferably at 20-30°C.
- a fermentation broth of Streptomyces clavuliqerus fermented in conventional manner was divided into four parts of 0.5 1.
- An amount of acetone equal to 11%, 25% and 117% compared to the sample volume was added to three broth samples respectively.
- the broth samples were then filtered separately.
- the time required for separation of the aqueous phase was found to decrease with increasing acetone concentration.
- the periods of filtration were 3600s, 2490s, 1200s and 800s respectively (see Table 1).
- the filtrates were contacted with ethyl acetate at a volume of 1.5 compared to the volume of the filtrate. All samples exhibited an intermediate layer, the thickness of which decreased with increasing amount of acetone added (1 cm, 0.5 cm, 0.1 cm and 0.03 cm, respectively) .
- the rate of static phase separation was dependent on the amount of acetone present. With 0, 11% and 25% acetone present in the initial broth sample, a relatively low phase separation was observed. With 117% of acetone present in the initial broth sample, a relatively fast separation was observed.
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Abstract
The present invention relates to a process for recovery of a fermentation product from an aqueous fermentation broth including the step of adding to the fermentation broth a water-miscible organic solvent so as to improve the filterability. Such a step is especially advantageous, for example, for improving recovery of clavulanic acid from a fermentation broth of a clavulanic acid producing microorganism.
Description
AN IMPROVED PROCESS TO RECOVER AN AQUEOUS FERMENTATION BROTH
The present invention relates to a process for recovery of a fermentation product from an aqueous fermentation broth and in particular to modification of the fermentation broth so as to improve filterability. This modification can, for instance, be advantageously applied to a fermentation broth of a clavulan¬ ic acid producing microorganism to improve the filterability of said fermentation broth and subsequent recovery of clavulanic acid, preferably as a salt.
Clavulanic acid is an antibiotic and a potent ?-lactamase inhibitor which finds widespread use as a therapeutic agent. It may be employed synergistically with penicillins and cephalos- porins, as described, for instance, in GB patent 1,508,977. It can be produced by fermentation of a clavulanic acid producing microorganism, for instance Streptomyces clavuliqerus, after which a recovery process is required. In published European patent application EP-A-387178, it .has been suggested to use flocculating agents to improve the filterability of the broth. The present invention provides a simpler and cheaper method to improve the filterability of the broth.
Surprisingly, it has been found possible to improve recov¬ ery of clavulanic acid from an aqueous fermentation broth by the simple addition of a water-miscible organic solvent to the fermentation broth so as to improve filterability.
Although the invention is described hereinafter with parti¬ cular reference to recovery of microbially-produced clavulanic acid, it will be appreciated that it is applicable to a broad range of fermentation products. Thus in one aspect, the present invention provides a process for the recovery of a fermentation product from a fermentation broth which includes the addition of a water-miscible organic solvent to the fermentation broth so as to improve the filterability of the same. Preferably, such a process is applied to recover clavulanic acid as a salt e.g. the potassium salt, from a fermentation
broth of a clavulanic acid producing microorganism, preferably Streptomyces clavuli erus.
Preferred organic solvents for addition to a fermentation broth for the purpose of a process according to the invention are methanol, ethanol, propanol, isopropanol, acetone and mixtures thereof. Acetone is particularly preferred.
It has been found that a suitable amount of water-miscible organic solvent which when added to a fermentation broth will improve filterability is between 10-200% by weight of the initial broth, preferably 50-175% and more preferably 100-140%.
Such a solvent addition may, for example, be advantageously applied for the preparation of potassium clavulanate in a procedure comprising:
- fermenting a strain of a clavulanic acid producing microorgan- ism to form a fermentation broth;
- adding an organic solvent which is miscible with water to the fermentation broth;
- separating the solid biomass of the fermentation broth from the aqueous solution part thereof by filtration; - distilling the solution part of the fermentation broth and optionally further concentrating the resulting aqueous sol¬ ution;
- extracting the resulting aqueous solution with an organic solvent which is immiscible or partly miscible with water and separating the organic and aqueous phases;
- evaporating the organic phase to concentrate the same;
- adsorbing impurities;
- adding an amine to the organic solution so as to precipitate clavulanic acid as an amine salt; - isolating the amine clavulanate by filtration;
- optionally recrystallizing;
- dissolving the amine clavulanate and reacting the same with a potassium ion source;
- isolating the potassium clavulanate by filtering and drying; and
- optionally recrystallizing and isolating the potassium clavulanate by filtering and drying.
Preferably, acetone is used as the organic solvent for addition to the fermentation broth. A suitable potassium salt for use in such a procedure is potassium 2-ethylhexanoate. Preferably, the amine N,N,N' ,N'-tetramethyl-l,2-diaminoethane (TMEDA) is employed, preferably under conditions such that mono- TMEDA clavulanate is formed, as described in PCT application 094/22873.
Addition of a water-miscible organic solvent to such a fermentation broth may not only improve the filterability of the broth, but may also improve the phase separation rate in the subsequent extraction step. It may additionally be found advan¬ tageous to supplement the organic solvent extraction system with a small amount of a de-emulsifier (see the example) .
An important additional factor favouring addition of acetone to a clavulanic acid-containing fermentation broth is the finding that clavulanic acid is more stable in aqueous solutions containing acetone.
It is possible to combine the addition of an organic solvent to the fermentation broth with gentle acidification, preferably to pH 3-5 by using mineral or organic acids, suitably hydrochloric acid, sulphuric acid or nitric acid. It is also permissible to add a flocculating agent, preferably a non-ionic or anionic agent.
In a preferred process for recovery of clavulanic acid in accordance with the present invention, a fermentation broth obtained in conventional manner by fermentation using S. clavulicrerus is treated with a water-miscible cold solvent, preferably acetone, at 10-200% by weight of the broth, preferab¬ ly 50-175% by weight and more preferably 100-140% by weight. The solvent added has a temperature of 0-30°C, preferably 3-6°C. The water-miscible organic solvent may contain up to 10% by weight of water. The resulting mixture is cooled, if necessary, prefer¬ ably down to 0-30°C, and most favourably down to 3-6°C.
The cold mixture is filtered, preferably after addition of a filter aid (e.g. Dicalite 4108) , to separate the biomass including precipitated solids from the aqueous phase, and the
resulting filtrate is kept at low temperature, preferably at 0-30°C, and most favourably at 3-6'C.
The filtrate is subsequently distilled in conventional manner to remove much of the water-miscible solvent. The tem- perature of the concentrate during evaporation is kept at 10-50°C, favourably 15-40°C, and most preferably at 20-30°C. After this step, the concentration of water-miscible solvent is in the range of 2.5-50% by weight of the total solution, prefer¬ ably 4-30%, and most favourably in the range of 10-20%. Preferably, the aqueous solution is further concentrated to increase the concentration of clavulanic acid. Preferred concentrations of clavulanic acid in the concentrate are 1-50 times the concentration of the broth after fermentation, and more favourably 4-10 times. The temperature of the concentrate during the evaporation is kept at 10-50°C, favourable 15-40°C and most preferably at 20-30°C.
The following example is offered only to illustrate the present invention, not to limit it.
Example
A fermentation broth of Streptomyces clavuliqerus fermented in conventional manner was divided into four parts of 0.5 1. An amount of acetone equal to 11%, 25% and 117% compared to the sample volume was added to three broth samples respectively. The broth samples were then filtered separately. The time required for separation of the aqueous phase was found to decrease with increasing acetone concentration. The periods of filtration were 3600s, 2490s, 1200s and 800s respectively (see Table 1).
Table 1
acetone added (%)* \ filtration period (s)
0 3600
11 2490
25 1200
117 800
* volume compared to broth sample volume
Following good mixing, the filtrates were contacted with ethyl acetate at a volume of 1.5 compared to the volume of the filtrate. All samples exhibited an intermediate layer, the thickness of which decreased with increasing amount of acetone added (1 cm, 0.5 cm, 0.1 cm and 0.03 cm, respectively) . The rate of static phase separation was dependent on the amount of acetone present. With 0, 11% and 25% acetone present in the initial broth sample, a relatively low phase separation was observed. With 117% of acetone present in the initial broth sample, a relatively fast separation was observed.
The addition of a drop of armogard (de-emulsifier) yielded a faster separation in the case of use of 25% acetone than in the case of use of 11% acetone. With no acetone present, the addition of an emulsifier had no significant effect on the phase separation rate.
Claims
1. A process to improve the recovery of a fermentation product from a fermentation broth which comprises the addition of a water-miscible organic solvent or a mixture of such organic solvents to the fermentation broth.
2. A process according to claim 1 wherein the organic solvent or a mixture of such organic solvents is added to a fermentation broth of a clavulanic acid producing microorganism.
3. A process as claimed in claim 1 or 2 wherein the amount of water-miscible organic solvent added to the fermentation broth is .10 to 200% by weight of the initial broth.
4. A process as claimed in claim 3 wherein the amount of water-miscible organic solvent added to the fermentation broth is 50 to 175% by weight of the initial broth.
5. A process as claimed in claim 4 wherein the amount of water-miscible organic solvent added'to the fermentation broth is 100 to 140% by weight of the initial broth.
6. A process as claimed in any one of claims 1 to 5 wherein said water-miscible organic solvent is selected from acetone, methanol, ethanol, propanol, isopropanol and mixtures thereof.
7. A process as claimed in any one of claims 2 to 6 wherein clavulanic acid is recovered as potassium clavulanate, which comprises:
- fermenting a strain of a clavulanic acid producing microorgan¬ ism to form a fermentation broth;
- adding a water-miscible organic solvent to the fermentation brot ; - separating the solid biomass of the fermentation broth from the aqueous solution part thereof by filtration; - distilling the solution part of the fermentation broth and optionally further concentrating the aqueous phase;
- extracting the resulting aqueous phase with an organic solvent which is immiscible or partly miscible with water and separat- ing the organic and aqueous phases;
- evaporating the organic phase to concentrate the same;
- adsorbing impurities;
- adding an amine to the organic solution so as to precipitate clavulanic acid as an amine salt; - isolating the amine clavulanate by filtration;
- optionally recrystallizing;
- dissolving the amine clavulanate and reacting the same with a potassium ion source;
- isolating the potassium clavulanate by filtering and drying; and
- optionally recrystallizing and isolating the potassium clavulanate by filtering and drying.
8. A process as claimed in any one of claims 1 to 7 wherein the water-miscible organic solvent added to the fermentation broth is acetone.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP93202968.9 | 1993-10-22 | ||
| EP93202968 | 1993-10-22 |
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| Publication Number | Publication Date |
|---|---|
| WO1995011295A1 true WO1995011295A1 (en) | 1995-04-27 |
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| Application Number | Title | Priority Date | Filing Date |
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| PCT/EP1994/003510 WO1995011295A1 (en) | 1993-10-22 | 1994-10-24 | An improved process to recover an aqueous fermentation broth |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997008175A1 (en) * | 1995-08-28 | 1997-03-06 | Lek Pharmaceutical & Chemical Co. Dd | Isolation of clavulanic acid from fermentation broth by ultrafiltration |
| US6207428B1 (en) | 1994-03-02 | 2001-03-27 | Lek Pharmaceutical & Chemical Co. D.D. | Process for the isolation of clavulanic acid and of pharmaceutically acceptable salts thereof from the fermentation broth of streptomyces sp. P 6621 FERM P 2804 |
| US6620325B2 (en) | 1996-09-11 | 2003-09-16 | Novartis Ag | Purification process for cyclosporin |
| CN108558909A (en) * | 2018-05-17 | 2018-09-21 | 国药集团威奇达药业有限公司 | The method of active ingredient in synthetical recovery clavulanic acid tert-butylamine salt crystalline mother solution |
Citations (6)
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|---|---|---|---|---|
| DE1923021A1 (en) * | 1968-05-10 | 1969-11-20 | Inst Francais Du Petrol | Process for the separation and purification of microorganisms from a fermentation mass |
| DD128051A3 (en) * | 1975-03-17 | 1977-10-26 | Walter Teich | METHOD FOR SEPARATING MICROORGANISMS FROM FERMENTO LEAVES |
| EP0137506A2 (en) * | 1983-10-11 | 1985-04-17 | The Dow Chemical Company | Extraction of teichmonycin a2 from whole culture fermentation broth |
| EP0182522A1 (en) * | 1984-10-27 | 1986-05-28 | Beecham Group p.l.c. | Preparation of clavulanic acid and its salts and esters |
| WO1991006552A1 (en) * | 1989-10-30 | 1991-05-16 | Eli Lilly And Company | A83543 recovery process |
| WO1992010580A1 (en) * | 1990-12-07 | 1992-06-25 | E.I. Du Pont De Nemours And Company | Natamycin recovery |
-
1994
- 1994-10-24 WO PCT/EP1994/003510 patent/WO1995011295A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1923021A1 (en) * | 1968-05-10 | 1969-11-20 | Inst Francais Du Petrol | Process for the separation and purification of microorganisms from a fermentation mass |
| DD128051A3 (en) * | 1975-03-17 | 1977-10-26 | Walter Teich | METHOD FOR SEPARATING MICROORGANISMS FROM FERMENTO LEAVES |
| EP0137506A2 (en) * | 1983-10-11 | 1985-04-17 | The Dow Chemical Company | Extraction of teichmonycin a2 from whole culture fermentation broth |
| EP0182522A1 (en) * | 1984-10-27 | 1986-05-28 | Beecham Group p.l.c. | Preparation of clavulanic acid and its salts and esters |
| WO1991006552A1 (en) * | 1989-10-30 | 1991-05-16 | Eli Lilly And Company | A83543 recovery process |
| WO1992010580A1 (en) * | 1990-12-07 | 1992-06-25 | E.I. Du Pont De Nemours And Company | Natamycin recovery |
Non-Patent Citations (1)
| Title |
|---|
| CHEMICAL ABSTRACTS, vol. 70, no. 5, 3 February 1969, Columbus, Ohio, US; abstract no. 18921f, page 1881; * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6207428B1 (en) | 1994-03-02 | 2001-03-27 | Lek Pharmaceutical & Chemical Co. D.D. | Process for the isolation of clavulanic acid and of pharmaceutically acceptable salts thereof from the fermentation broth of streptomyces sp. P 6621 FERM P 2804 |
| US6566106B2 (en) | 1994-03-02 | 2003-05-20 | Lek Pharmaceutical & Chemical Co., D.D. | Process for the isolation of clavulanic acid and of pharmaceutically acceptable salts thereof |
| WO1997008175A1 (en) * | 1995-08-28 | 1997-03-06 | Lek Pharmaceutical & Chemical Co. Dd | Isolation of clavulanic acid from fermentation broth by ultrafiltration |
| US6127358A (en) * | 1995-08-28 | 2000-10-03 | Urquhart-Dykes & Lord | Isolation of clavulanic acid from fermentation broth by ultrafiltration |
| US6274575B1 (en) | 1995-08-28 | 2001-08-14 | Lek Pharmaceutical And Chemical Co. D. D. | Isolation of clavulanic acid from fermentation broth by ultrafiltration |
| US6620325B2 (en) | 1996-09-11 | 2003-09-16 | Novartis Ag | Purification process for cyclosporin |
| US7148346B2 (en) | 1996-09-11 | 2006-12-12 | Novartis Ag | Purification process |
| CN108558909A (en) * | 2018-05-17 | 2018-09-21 | 国药集团威奇达药业有限公司 | The method of active ingredient in synthetical recovery clavulanic acid tert-butylamine salt crystalline mother solution |
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